Prevention of ischemia-reperfusion-induced hepatic microcirculatory disruption by inhibiting stellate cell contraction using rock inhibitor

BACKGROUND: We demonstrated that hepatic stellate cells (HSCs) isolated from rat livers exposed to warm ischemia are significantly contractile when compared with HSCs from intact rat livers. This suggests that ischemia-reperfusion (IR)-induced impairment of sinusoidal microcirculation results, at least in part, from contraction of HSCs. METHODS: Rho-associated coiled-coil forming protein serine/threonine kinase (ROCK) is one of the key regulators of HSCs motility. Therefore we investigated whether Y-27632, a p160ROCK-specific inhibitor, has beneficial effects on warm IR injury in an in vivo rat partial liver IR model and a rat orthotopic liver transplantation model. RESULTS: After reperfusion following 90 min of warm ischemia, livers in untreated control rats had persistent congestion and impaired mitochondrial respiration, as demonstrated by increasing deoxy-hemoglobin and reduced cytochrome oxidase contents in the hepatic tissues using in vivo near-infrared spectroscopy. Serum levels of transaminase and endothelin (ET)-1 in these rats were markedly increased 1 hr after reperfusion. In contrast, when Y-27632 (3-30 mg/kg) was administered orally, hepatic tissue contents of deoxy-hemoglobin and cytochrome oxidase rapidly normalized. In such animals, the elevation of serum transaminase levels, but not that of ET-1 levels, was significantly suppressed. This is consistent with in vitro data demonstrating that Y-27632 causes HSCs to undergo relaxation even in the presence of ET-1. Moreover, in a rat orthotopic liver transplantation model, Y-27632 pretreatment dramatically improved the survival of recipients with liver grafts subjected to 45 min of warm ischemia. CONCLUSIONS: Y-27632 attenuates IR-induced hepatic microcirculation disruption by inhibiting contraction of HSCs.     

Therapeutic significance of Y-27632, a Rho-kinase inhibitor,on the established liver fibrosis

BACKGROUND: Recently, we demonstrated that Y-27632, a Rho-kinase inhibitor, inhibited hepatic stellate cells (HSCs) activation in terms of cellular morphology, improved the progression of carbon tetrachloride (CCl(4))-induced rat liver fibrosis. The objective of the present study was to investigate the effects of Y-27632 on the established liver fibrosis. METHODS AND METHODS: Liver cirrhosis was induced by intragastric administration of CCl(4) once a week for 12 weeks. After the first 6 weeks of CCl(4) injection, Y-27632 (30 mg/kg body weight) or saline was continuously administered to the rats via an intraperitoneally implanted osmotic pump during the final 6 weeks of CCl(4) injection. Two days after the last CCl(4) injection, 70% hepatectomy was performed. RESULTS: Y-27632 prevented the development of CCl(4)-induced liver fibrosis and improved the fibrotic changes, hydroxyproline content, and serum hyaluronic acid level in the liver. Moreover, Y-27632 reduced the number of smooth muscle alpha-actin- and transforming growth factor beta1-positive cells, and inhibited the expression of Na(+)/Ca(2+) exchanger mRNA which was reported to be an indicator of HSCs activation and liver fibrosis. Further, the Y-27632-treated group showed markedly increased survival rate after hepatectomy. CONCLUSIONS:These findings indicated that Y-27632 may be useful therapeutically in liver cirrhosis.     

Rho-Associated Kinase Inhibitor Reduces Tumor Recurrence After Liver Transplantation in a Rat Hepatoma Model

Tumor recurrence after liver transplantation still remains a significant problem in patients with hepatocellular carcinoma. The small GTPase Rho/Rho-associated kinase (ROCK) pathway is involved in the motility and invasiveness of cancer cells. We investigated whether tacrolimus activated the Rho/ROCK signal pathway to promote the invasiveness of rat hepatocellular carcinoma cells. We also investigated whether the ROCK inhibitor Y-27632 suppressed tumor recurrence after experimental liver transplantation in a rat hepatocellular carcinoma model. Orthotopic liver transplantation was performed in hepatocellular carcinoma cell line McA-RH7777-bearing rats. Tacrolimus was administered to liver transplant rats and these rats were divided into two groups: the Y-27632-treated (10 mg/kg, for 28 days) group and the Y-27632-untreated group. Tacrolimus enhanced the cancer cell migration and stimulated phosphorylation of the myosin light chain (MLC), a downstream effector of Rho/ROCK signaling. Y-27632 suppressed the cancer cell migration and tacrolimus-induced MLC phosphorylation. Suppression of tumor recurrence after liver transplantation and significant prolongation of survival were observed in the Y-27632-treated rats in comparison with theY-27632-untreated rats. Tacrolimus stimulates the Rho/ROCK signal pathway to enhance the invasiveness of hepatocellular carcinoma, and the ROCK inhibitor Y-27632 can be used as a new antimetastatic agent for the prevention of tumor recurrence after liver transplantation.     

Serelaxin induces Notch1 signaling and alleviates hepatocellular damage in orthotopic liver transplantation

Liver ischemia‐reperfusion injury (IRI) represents a risk factor for early graft dysfunction and an obstacle to expanding donor pool in orthotopic liver transplantation (OLT). We have reported on the crucial role of macrophage Notch1 signaling in mouse warm hepatic IRI model. However, its clinical relevance or therapeutic potential remain unknown. Here, we used Serelaxin (SER), to verify Notch1 induction and putative hepatoprotective function in ischemia‐reperfusion–stressed OLT. C57BL/6 mouse livers subjected to extended (18‐hour) cold storage were transplanted to syngeneic recipients. SER treatment at reperfusion ameliorated IRI, improved post‐OLT survival, decreased neutrophil/macrophage infiltration, and suppressed proinflammatory cytokine programs, while simultaneously increasing Notch intracellular domain (NICD) and hairy and enhancer of split 1 (Hes1) target genes. In bone marrow–derived macrophage cultures, SER suppressed proinflammatory while enhancing antiinflammatory gene expression concomitantly with increased NICD and Hes1. Hepatic biopsies from 21 adult primary liver transplant patients (2 hours postreperfusion) were divided into low‐NICD (n = 11) and high‐NICD (n = 10) expression groups (western blots). Consistent with our murine findings, human livers characterized by high NICD were relatively IRI resistant, as shown by serum alanine aminotransferase (ALT) levels at day 1 post‐OLT. Our study documents the efficacy of SER–Notch1 signaling in mouse OLT and highlights the protective function of Notch1 in liver transplant patients.     

A highly selective inhibitor of Rho-associated coiled-coil forming protein kinase, Y-27632, prolongs cardiac allograft survival of the BALB/c-to-C3H/He mouse model.

BACKGROUND: Current studies provide evidence that a small G protein, RhoAp21, and its target protein, Rho-associated coiled-coil forming protein kinase (ROCK), regulate not only cell shape but also cell migration. However, contribution of Rho/ROCK signaling to graft rejection is unknown. The purpose of this study was to evaluate the inhibitory effect of Y-27632, a highly selective ROCK inhibitor, on rejection of heterotopic cardiac transplantation in mice. METHODS: BALB/c (H-2(d)) hearts were transplanted into C3H/He (H-2(k)) as allografts that were full histoincompatibility combinations. The recipients received several doses of Y-27632, commencing 1 day before cardiac transplantation until rejection. We used immunohistochemical study to detect the expression of myocardial intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1), and we immunoenzymatically measured serum interleukin (IL)-6. Furthermore, we evaluated cardiac allograft vasculopathy treated with either FK506 or Y-27632 at Day 100. RESULTS: The Y-27632-treated (2 mg/kg/day) allografts prolonged the mean survival time (49.6 +/- 10.1 days, n = 12) as compared with the untreated allografts (8.1 +/- 0.4 days, n = 7, p < 0.001). Histologic examinations of the Y-27632-treated allografts at Day 7 showed greatly reduced leukocyte infiltration compared with the untreated allografts. The Y-27632-treated allografts revealed faint expression of myocardial ICAM-1 and VCAM-1 at Day 7. The serum IL-6 levels also decreased in the Y-27632-treated mice. In the long-surviving Y-27632-treated allografts at Day 100, we saw neither active rejection nor apparent thickening of vascular intima. CONCLUSION: Our results suggest that ROCK plays a major role in cardiac rejection in the BALB/c-to-C3H/He mouse model. Inhibition of this Rho/ROCK signaling may be an alternative therapeutic option for managing acute and chronic rejection.     

A novel inhibitor of Rho-associated protein kinase,Y-27632, ameliorates hepatic ischemia and reperfusion injury in rats

BACKGROUND: A Rho-ROCK signal system induces vascular contraction and neutrophil migration, both of which are characteristic features found with ischemia and reperfusion injury of the liver. We tested our hypothesis that a novel ROCK I inhibitor, Y-27632, attenuates hepatic ischemia and reperfusion injury. METHODS: Rats underwent 70% partial hepatic ischemia for 120 minutes and subsequent reperfusion. Y-27632 of 10mg/kg was given orally 1 hour before ischemia, while distilled water was given to the control animals. One week animal survival, systemic hemodynamics, hepatic tissue blood flow, liver function tests, plasma endothelin-1, serum hyaluronic acid levels, myeloperoxidase activity and malondialdehyde level in liver tissue, membrane attack complex-1 and intracellular adhesion molecule-1 staining, and histological architecture were analyzed. RESULTS: Y-27632 prolonged 1-week animal survival from 25% of untreated animals to 75% accompanied with significant amelioration of hepatic tissue blood flow, liver function tests and histological architecture without any adverse effects on systemic hemodynamics. In addition, plasma endothelin-1 and serum hyaluronic acid levels decreased markedly compared to the control, concomitant with remarkable suppression of membrane attack complex-1 stain positive neutrophils infiltration, myeloperoxidase activity and malondialdehyde level. CONCLUSION: Present study suggests that activation of a Rho-ROCK signal system is associated with ischemia and reperfusion injury of the liver, and that Y-27632 may be an attractive agent for application in major liver resection using temporary inflow occlusion and hepatic preservation.     

Diannexin, a Novel Annexin V Homodimer, Protects Rat Liver Transplants Against Cold Ischemia-Reperfusion Injury

Ischemia/reperfusion injury (IRI) remains an important problem in clinical transplantation. Following ischemia, phosphatidylserine (PS) translocates to surfaces of endothelial cells (ECs) and promotes the early attachment of leukocytes/platelets, impairing microvascular blood flow. Diannexin, a 73 KD homodimer of human annexin V, binds to PS, prevents attachment of leukocytes/platelets to EC, and maintains sinusoidal blood flow. This study analyzes whether Diannexin treatment can prevent cold IRI in liver transplantation. Rat livers were stored at 4 degrees C in UW solution for 24 h, and then transplanted orthotopically (OLT) into syngeneic recipients. Diannexin (200 microg/kg) was infused into: (i) donor livers after recovering and before reperfusion, (ii) OLT recipients at reperfusion and day +2. Controls consisted of untreated OLTs. Both Diannexin regimens increased OLT survival from 40% to 100%, depressed sALT levels, and decreased hepatic histological injury. Diannexin treatment decreased TNF-alpha, IL-1beta, IP-10 expression, diminished expression of P-selectin, endothelial ICAM-1, and attenuated OLT infiltration by macrophages, CD4 cells and PMNs. Diannexin increased expression of HO-1/Bcl-2/Bcl-xl, and reduced Caspase-3/TUNEL+ apoptotic cells. Thus, by modulating leukocyte/platelet trafficking and EC activation in OLTs, Diannexin suppressed vascular inflammatory responses and decreased apoptosis. Diannexin deserves further exploration as a novel agent to attenuate IRI, and thereby improve OLT function/increase organ donor pool.     

Analysis of clinical variables of donors and recipients with respect to short-term graft outcome in human liver transplantation

Donor and recipient factors are closely associated with graft survival after orthotopic liver transplantation (OLT). This study was performed to analyze clinical characteristics of recipients and donors, which affect 30-day graft loss after OLT. MATERIALS AND METHODS: One hundred eighty-six livers from heart-beating donors were accepted between May 1997 and June 1998 at the University of Pittsburgh Medical Center. Donor variables that were analyzed included age, sex, cold ischemia time (CIT), warm ischemia time (WIT), imported versus local procurement, cardiopulmonary arrest, serum sodium level, and dopamine dose. The recipient characteristics included native liver disease and UNOS status. Two-sided Fisher exact test and stepwise logistic regression were used for univariate and multivariate analyses. P-values < .05 were considered statistically significant. RESULTS: Twenty-eight grafts (15.1%) were lost within 30 days of OLT. The following factors were found to adversely affect graft survival: donor sodium > 155 mEq/L (P = .002); CIT > 12 hours (P = .002); WIT > 45 minutes (P = .002); and imported liver graft (P = .048). Logistic regression revealed that donor sodium (odds ratio, 3.03; 95% CI, 1.05 to 8.74), CIT (OR 1.20; 95% CI 1.05 to 1.38), WIT (OR 1.06; 95% CI 1.01 to 1.09) were independent predictors of early graft loss. CONCLUSION: Donor hypernatremia as well as warm and cold ischemia times independently affect graft outcomes in the early postoperative period after OLT. Avoidance of long preservation and correction of donor sodium level are recommended to optimize results and survival in OLT.     

c-Jun N-terminal kinase mediates hepatic injury after rat liver transplantation

BACKGROUND: Orthotopic liver transplantation (OLT) requires cold ischemic storage followed by warm reperfusion. Although c-Jun N-terminal kinase (JNK) is rapidly activated after OLT, the functional consequences of JNK activation are unknown. The aim of this study was to address the role of JNK after OLT using the selective JNK inhibitor CC-401. METHODS: Donors, recipients, or stored liver explants were treated with vehicle or JNK inhibitor before OLT by an arterialized two-cuff method with 40 hours of cold storage. Recipients were assessed for 30-day survival, and graft injury was assessed over time by hepatic histology, serum transaminases, caspase 3 activation, cytosolic cytochrome c, and lipid peroxidation. RESULTS: Survival after OLT increased after donor plus storage and storage only treatment with JNK inhibitor (P<0.05). Treatment of recipient only did not improve survival. Increased survival correlated with improved hepatic histology and serum aspartate aminotransferase levels. JNK inhibition significantly decreased nonparenchymal cell killing at 60 minutes after reperfusion (P<0.05) and pericentral necrosis at 8 hours after reperfusion (P<0.01). JNK inhibition decreased cytochrome c release, caspase 3 activation (P<0.05), and lipid peroxidation (P<0.05). JNK inhibition also transiently blocked phosphorylation of c-Jun at 60 minutes after reperfusion (P<0.05) without affecting other MAPK signaling, including p-38 and Erk activation. CONCLUSIONS: JNK inhibition decreases hepatic necrosis and apoptosis after OLT, suggesting that JNK activation promotes cell death by both pathways. Inhibition of JNK may be a new therapeutic strategy to prevent liver injury after transplantation.     

P-Selectin Glycoprotein Ligand-1 (rPSGL-Ig)-Mediated Blockade of CD62 Selectin Molecules Protects Rat Steatotic Liver Grafts from Ischemia/Reperfusion Injury

We examined the effects of early blockade of CD62 selectin-mediated adhesive interactions in steatotic rat liver models of ex vivo cold ischemia followed by reperfusion or transplantation by administration of P-selectin glycoprotein ligand-1 (rPSGL-Ig). In the model of cold ischemia/reperfusion, livers pretreated ex vivo with rPSGL-Ig at harvesting from obese Zucker rats showed significantly decreased portal resistance, increased bile production, and diminished hepatic endothelial neutrophil infiltration, as compared with untreated controls. Pretreatment of fatty livers with rPSGL-Ig prior to transplantation extended the survival of lean Zucker rat recipients from 40% to 90%. This effect correlated with significantly improved liver function, depressed neutrophil activity, and decreased histologic features of hepatocyte injury. Intragraft expression of CD62 P-selectin was similar in both recipient groups. rPSGL-Ig treatment decreased intragraft infiltration by CD3/CD25 cells, diminished expression of pro-inflammatory TNFalpha, IL-6, iNOS, IL-2 and IFN-gamma, without significantly affecting mRNA levels coding for anti-inflammatory IL-4. Thus, rPSGL-Ig blockade of CD62-mediated adhesive interactions protects against severe ischemia/reperfusion injury suffered otherwise by steatotic rat livers. These findings document the potential utility of rPSGL-Ig in increasing the transplant donor pool through modulation of marginal steatotic livers.     

Coagulation disorders during orthotopic liver transplantation

Coagulation disorders have been noted during orthotopic liver transplantation (OLT) especially just after reperfusion of the grafted liver. This study was undertaken to clarify the coagulation disorders following reperfusion of the liver graft. OLT was carried out in adult mongrel dogs using a cuff technique. Fresh and 24-hour preserved livers were grafted. Platelet count (P1), activated partial thromboplastin time (A-PTT), prothrombin time (PT) and plasma fibrinogen levels (Fng) were measured before and after OLT. Next perfusate obtained from fresh livers or preserved livers for 24-hours or 48-hours was determined for their ability of inducing coagulation disorders when infused in untreated dogs, and was also tested for their activity of platelet aggregation, tissue thromboplastin (F-III), and plasminogen activator (PA). All dogs which received preserved livers showed a marked coagulation disorders including a decrease in P1 and Fng, and prolongation of A-PTT and PT, but the dogs with fresh liver grafts did not. Infusion of the perfusate collected from a perfusion of the preserved liver induced similar coagulation disorders in untreated dogs. The perfusate obtained from the preserved liver showed significant increased F-III activity as compared with that from fresh liver. On the other hand, neither direct platelet aggregation activity nor PA activity was seen or very low if any. These results indicate that F-III liberated from a damaged liver is responsible for the coagulation disorders after reperfusion of the graft in liver transplantation.     

Predisposing factors for biliary complications following liver transplantation

INTRODUCTION: Biliary complications remain a major cause of morbidity and mortality in patients following liver transplantation. We sought to identify possible risk factors predisposing to biliary complications after OLT using duct-to-duct biliary reconstruction. MATERIALS AND METHODS: We retrospectively reviewed 5 years of prospectively collected donor and recipient data between April 1999 and April 2004. We evaluated the presence of biliary complications, donor and recipient age, cold ischemia time, hepatic artery thrombosis, non-heart-beating donor (NHBD), and graft steatosis (>30%). The results were compared with a control group of OLT patients without biliary complications. RESULTS: Among 173 OLT recipients, biliary complications occurred in 28 patients (16.2%), of whom 12 were leaks, 15 strictures, and 1 a nonanastomotic intrahepatic stricture. The mortality following biliary complications was 11%, compared to 6% in the control group. CONCLUSION: Biliary complications remain a persistent problem in OLT. Analysis of risk factors identified hepatic artery thrombosis and steatosis as predisposing factors. With greater experience, NHBD livers may also prove to be at greater risk of biliary complications.     

Sotrastaurin, a protein kinase C inhibitor, ameliorates ischemia and reperfusion injury in rat orthotopic liver transplantation

Sotraustaurin (STN), a small molecule, targeted protein kinase C (PKC) inhibitor that prevents T-lymphocyte activation via a calcineurin-independent pathway, is currently being tested in Phase II renal and liver transplantation clinical trials. We have documented the key role of activated T cells in the inflammation cascade leading to liver ischemia/reperfusion injury (IRI). This study explores putative cytoprotective functions of STN in a clinically relevant rat model of hepatic cold ischemia followed by orthotopic liver transplantation (OLT). Livers from Sprague-Dawley rats were stored for 30 h at 4°C in UW solution, and then transplanted to syngeneic recipients. STN treatment of liver donors/recipients or recipients only prolonged OLT survival to >90% (vs. 40% in controls), decreased hepatocellular damage and improved histological features of IRI. STN treatment decreased activation of T cells, and diminished macrophage/neutrophil accumulation in OLTs. These beneficial effects were accompanied by diminished apoptosis, NF-κB/ERK signaling, depressed proapoptotic cleaved caspase-3, yet upregulated antiapoptotic Bcl-2/Bcl-xl and hepatic cell proliferation. In vitro, STN decreased PKCθ/IκBα activation and IL-2/IFN-γ production in ConA-stimulated spleen T cells, and diminished TNF-α/IL-1β in macrophage-T cell cocultures. This study documents positive effects of STN on liver IRI in OLT rat model that may translate as an additional benefit of STN in clinical liver transplantation.     

Ischemic cholangiopathy after liver transplantation from controlled non-heart-beating donors-a single-center experience

BACKGROUND: Previous reports have shown that livers from controlled non-heart-beating-donors (NHBD) are associated with higher rates of primary failure and ischemic cholangiopathy of orthotopic liver transplantation (OLT) as a complication of the prolonged warm ischemia. METHODS: This retrospective review of activities from 1999 to 2006 examined donor characteristics of age, liver function tests, warm ischemic time before (1WITa) and after cardiac arrest (1WITb), cold ischemic time (CIT) and transplant results. RESULTS: Eleven NHBD retrieved livers were transplanted from "ideal" donors except for one elderly donor (73 years). Of the 11 recipients, 3 developed biliary cholangiopathy (27%). There were no episodes of primary graft nonfunction, but one recipient displayed primary graft dysfunction. Two recipients died: one due to biliary complications with sepsis (long CIT >10 hours, fatty liver), and the other due to aspiration pneumonia and hypoxic brain damage with normal liver function. One recipient required retransplantation owing to ischemic cholangiopathy (1WITb 45 min) at 6 months after OLT with a good result. The other eight recipients are alive (observation period 72 to 14 months) including six with normal liver function, one with biopsy-proven biliary ischemia and one with recurrent primary sclerosing cholangitis without biliary ischemic changes on biopsy. Among 164 heart-beating donors recipients transplanted in the same period, biliary complications occurred in 27 patients (16%), of whom 12 were leaks and 15 anastomotic strictures. CONCLUSION: NHBD were a good source for livers with reasonable early results. To avoid late complications especially ischemic cholangiopathy, caution is urged with the use of these organs as well as strict donor and ischemic time criteria.     

Reduction of hepatic ischemia/reperfusion-induced injury by a specific ROCK/Rho kinase inhibitor Y-27632

BACKGROUND: The low-molecular-weight GTPase Rho is known to act as a molecular switch by activating several downstream effectors, one of which is Rho-associated coiled-coil forming protein kinase (ROCK). ROCK/Rho kinase mediates cytoskeleton-dependent cell functions, such as actomyosin-based smooth muscle contraction and integrin-mediated cell adhesion. A specific inhibitor of ROCK/Rho kinase, Y-27632, was recently developed. The present study examined whether Y-27632 could provide a beneficial effect on hepatic ischemia/reperfusion (I/R)-induced injury through the attenuation of microcirculatory disturbance. MATERIALS AND METHODS: In male Sprague-Dawley rats, normothermic partial ischemia was induced by clamping the hepatic pedicle to the left and median lobes for 90 min, followed by 2 h of reperfusion. In the treatment group, Y-27632 was intravenously administered prior to ischemic insult. Hepatic microcirculation was investigated by using intravital fluorescence microscopy. Liver enzyme release and histological changes of the liver tissue were also evaluated. RESULTS: Y-27632 significantly improved sinusoidal perfusion and reduced the number of leukocytes sticking in hepatic sinusoids and adhering in postsinusoidal venules. The postischemic narrowing of both sinusoids and postsinusoidal venules was also markedly suppressed. Consequently, liver enzyme release was reduced and postischemic histological damage was suppressed. CONCLUSIONS: A specific ROCK/Rho kinase inhibitor, Y-27632, was useful to alleviate hepatic I/R-induced injury through ameliorating postischemic microcirculation. The administration of Y-27632 may be a novel strategy for conquering hepatic I/R-induced injury.     

N-Acetylcysteine failed to improve early microcirculatory alterations of the rat liver after transplantation

The application of radical scavengers reduces reperfusion injury of liver grafts despite the natural occurrence of cellular defense mechanisms enabling the cell to tolerate moderate oxidant stress without further cell damage. The glutathione peroxidase mechanism of the liver serves to reduce hydroxyl radical-induced lipid peroxidation by releasing reduced glutathione from intracellular stores. There is evidence that the application of cysteine-providing aminoacids for glutathione synthesis could maintain or even increase liver glutathione. Therefore, the purpose of this study was to evaluate the effect of N-acetylcysteine (NAC) on oxidative stress-induced reperfusion injury after liver transplantation. This was done by applying intravital microscopy. Livers from female Sprague-Dawley rats weighing 220–260 g were stored for 20 h in University of Wisconsin (UW) solution and transplanted orthotopically using the cuff technique. Donors were given 150 mg/kg body weight NAC i. v. or placebo in a blind, random fashion 6 h prior to harvesting, followed by two injections of 50 mg/kg body weight, 4 and 2 h before explantation. In additional experimental groups, recipients were given a bolus of 83 mg/kg body weight NAC or placebo at the beginning of the recipient operations, 1 min prior to reperfusion, and 60 min after surgery. Ninety minutes after transplantation, intravital microscopy was applied and five liver lobules were recorded for 30 s after injection of acridine orange, a fluorescent leukocyte marker. Sinusoidal perfusion, sinusoidal width, and leukocyte adhesion, as well as reduced and oxidized glutathione, were determined in all livers. Neither microcirculatory disturbance nor leukocyte adhesion was less, nor was the liver glutathione in the recipient groups pretreated or treated with NAC greater than that in rats receiving the placebo. Moreover, liver glutathione was significantly decreased in livers from donors pretreated with NAC. In conclusion, the application of NAC as a pretreatment for donors and as treatment for recipients, respectively, failed to reduce early microvascular failure after liver transplantation.     

Ischemic preconditioning of cadaver donor livers protects allografts following transplantation

BACKGROUND: Ischemic preconditioning (IP) has been shown in animal models to protect livers against ischemia/reperfusion injury. The aim of this clinical study is to investigate whether IP of cadaver livers prior to retrieval confers protection on the allografts. METHODS: Cadaveric donor livers were subjected to IP prior to retrieval by clamping of the hepatic pedicle for 10 min followed by reperfusion. Biopsies were obtained from the preconditioned (n=9) and control nonpreconditioned (n=14) liver transplants prior to and 2 hr following reperfusion. Cryosections were stained with antibodies against neutrophils and platelets. RESULTS: IP livers were associated with significantly lower serum levels of aspartate aminotransferase (240+/-98 IU/L vs. 382+/-163 IU/L; P>0.016) and lactate (0.81+/-0.07 mmol/L vs. 1.58+/-0.9 mmol/L; P>0.018) 24 hr following transplantation. Furthermore, recipients of IP livers spent a significantly shorter time in the intensive care unit following transplantation compared to those given nonpreconditioned allografts (1 vs. 2.8+/-1.6 days; P=0.0008). Increases in neutrophil infiltration were detected in 6/14 (43%; P=0.022) and in CD41 deposition in 5/14 (36%; P=0.042) of nonpreconditioned livers. However, none of the IP allografts showed any change in the levels of platelets or neutrophil infiltration following transplantation. CONCLUSION: IP is an effective method of protecting cadaver donor allografts from cold ischemia and subsequent reperfusion injury. IP is also associated with a reduction in the nonspecific inflammatory response.     

A caspase inhibitor, IDN-6556, ameliorates early hepatic injury in an ex vivo rat model of warm and cold ischemia.

This study examined the efficacy of the caspase inhibitor, IDN-6556, in a rat model of liver ischemia-reperfusion injury. Livers from male Sprague-Dawley rats were reperfused for 120 minutes after 24 hours of 4 degrees C cold storage in University of Wisconsin solution. Portal blood flow measurements estimated sinusoidal resistance, and bile production, alanine aminotransferase activities, and Suzuki scores were evaluated as parameters of hepatocyte/liver injury. Treated livers were exposed to 25 or 50 microM of IDN-6556 in University of Wisconsin storage solution and/or the perfusate. All treatment regimens with IDN-6556 significantly improved portal blood flow measured at 120 minutes, and significant improvements were seen as early as 30 minutes when inhibitor was also present in the perfusate (P < 0.01). All treatment groups with IDN-6556 significantly increased bile production by 3-4-fold compared with controls (P < 0.01), and reductions in alanine aminotransferase activities were seen within 90 minutes of reperfusion (P < 0.05). These data were confirmed by improved Suzuki scores (less sinusoidal congestion, necrosis, and vacuolization) in all treated groups. Livers from the IDN-6556-treated groups had markedly reduced caspase activities and TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling)-positive cells, suggesting reductions in apoptosis. IDN-6556 present in cold storage media ameliorated liver injury due to cold ischemia and reperfusion injury and may be a rational therapeutic approach to reduce the risk of liver ischemia in the clinical setting.